The invention relates to a vector which can be replicated in cultures of unicellular organisms, this vector containing a gene coding for a eucaryotic protein having the biological activity of a membrane receptor and interacting with a regulatory proteinxe2x80x94called G proteinxe2x80x94capable of binding molecules of guanosine triphosphate (GTP).
The invention also relates to unicellular organisms transformed by genes coding for these membrane receptors and bearing at their surface peptide sequences recognized by specific ligands of the membrane receptors.
The invention also relates to kits for the identification of mammalian proteins which have properties making it possible to verify whether they belong to a family of mammalian membrane receptors or, conversely, to study the degree of affinity of certain substances for specific membrane receptors.
Generally speaking, the G proteins are proteins having the capacity to interpose themselves structurally and functionally between receptors and enzymes catalyzing the production of intracellular mediators (such as adenylate cyclase, guanylate cyclase, the phospholipases, the kinases) or between receptors and ionic channels, the controlled opening of which leads to a flux of ions (such as calcium, potassium, sodium and hydrogen ions) into the cell.
These proteins have transduction and coupling functions and are characterized by a monomeric structure composed of three protein subunits alpha, beta, gamma. Different G proteins can be distinguished on the basis of variations observed in their alpha subunit and sometimes in their beta subunit. These G proteins have been described in detail in the article by Allen M. Spiegel (21).
Membrane receptors of mammalian cells normally coupled to a G protein have been identified by cloning, supplemented by the analysis of their sequence in the case of some of them. These receptors include regions exposed at the surface of the host cells which have specific affinities for a large variety of ligands, hormones, neurotransmitters and other sensory stimulants capable of transmitting signals to the interior of these cells via the corresponding receptors and the G proteins.
Receptors of interest in the context of the invention are, in particular, the following receptors:
xcex2-adrenergic receptors: (xcex21 avian receptor, xcex21 and xcex22 mammalian receptors), receptors for glucagon, the adrenocorticotropic hormone, the parathyroid hormone (PTH), the thyroid-stimulating hormone, the growth hormone releasing factor, prostaglandin E, D1 dopaminergic receptors, the intestinal vasoactive peptide receptor, the mucarinic receptors M1 to M6, the S1 and S2 serotonin receptors, the D2 dopaminergic receptor, the receptors for the neuropeptide substance K, vasopressin, somatostatin, bombesin, cholecystokinin (CCK), light receptors, the receptor for the chemotactic peptide Met-Leu-Phe, the xcex1 adrenergic receptors (xcex11, xcex12), the H1 histamine receptor, the opioid receptor (enkephalins, endorphins), the receptors for the neuropeptide substance P, angiotensin II, prostaglandin, the corticotropic hormone releasing factor, bradykinin, secretin and the olfactory receptors.
A preferred family of receptors according to the invention which possess genetic and molecular properties which are essentially common to all of them, comprises xcex21, xcex22, xcex11 and xcex12-adrenergic receptors (1) (2) (3) (4), muscarinic receptors belonging to the subtypes M1 to M4 (5) (6) and the receptor for the neuropeptide known as xe2x80x9csubstance Kxe2x80x9d (7). These receptors appear to possess a common structural organization within the plasma membrane, usually characterized by seven hydrophobic transmembrane segments between which are inserted extra- and intra-cellular loops, by an extracellular amino-terminal region and by a cytoplasmic carboxyl-terminal region.
Several genes coding respectively for some of these receptors have been made capable of expressing themselves after their introduction into cultures of mammalian cells belonging to foreign species. The operations of transformation necessary for the expression of these genes in mammalian cells belonging to foreign species are quite complex and cannot easily be discussed in terms of the expression of other receptors.
The aim of the invention is to furnish procedures and agents making it easy to identify and study new membrane receptors exhibiting structural characteristics in common with the membrane receptors of the type indicated above. Conversely, the aim of the invention is to facilitate the study of the possible degree of affinity of molecules capable of behaving as ligands with respect to these membrane receptors.
In particular, the invention relates to a vector, which can replicate in a culture of a host cell, containing a nucleotide insert coding for a sequence of amino acids contained in a mammalian protein and having the biological activity of a membrane receptor, this vector being characterized in that the insert is placed under the control of a sequence included in this vector allowing the expression of this insert in the host cell, and in particular including a promoter recognized by the polymerases of this unicellular host, and in that this insert codes for a sequence of amino acids contained in a protein possessing structural elements in common with the above-mentioned membrane receptors, in particular the xcex1- or xcex2-adrenergic receptors, the muscarinic receptors, the neuropeptide receptors, in particular for xe2x80x9csubstance Kxe2x80x9d.
The invention is the result of the discovery that placing a sequence of nucleotides coding for a mammalian membrane receptor under the control of a promoter recognized by the polymerases of unicellular host under conditions which allow the expression of this sequence of nucleotides within the unicellular organism also makes possible, when this expression occurs, the transport of the expressed product into the membranes of the unicellular host, and that this expression is also accompanied by the exposure, also at the exterior of these unicellular hosts, of sites characteristic of the membrane receptors which appear at the surface of the cells of mammalian origin, and do so in a spatial configuration that is conserved at the molecular level such that these sites can be recognized by the same ligands.
By xe2x80x9cunicellular hostxe2x80x9d is understood any organisms capable of being maintained in culture in the absence of cellular differentiation. The unicellular organism of choice is constituted by Escherichia coli. However, other microorganisms can be used with equal facility, provided of course that vectors are known for each of them, especially of the plasmid type, which are capable of self replication and nucleotide sequences which can be inserted into these vectors and which are capable, when they are followed in these vectors by an insert coding for a polypeptide having the properties of a membrane receptor under the conditions indicated above, of insuring the expression of this insert in the selected organisms and its transport into the membranes of these unicellular hosts.
In the case where the expression is to be carried out in E. coli, the sequence directing the expression of the insert may include, for example, the elements of regulation, in particular the promoter of the lactose operon or the tryptophan operon.
Other unicellular microorganisms capable of being used and, consequently, other vectors which can themselves be modified by a sequence capable of directing the expression of a membrane receptor or similar structure in these unicellular organisms are indicated in the table below:
The above-mentioned organisms and the corresponding, modified vectors advantageously lend themselves to the expression of genetic sequences coding for molecules having structural properties in common with mammalian membrane receptors already known, and to their transport to the membrane of these unicellular hosts under the conditions indicated above.
As examples of receptors likely to be introduced and expressed into unicellular organisms, in particular E. coli. mention should be made of:
the different xcex2-adrenergic and muscarinic receptors described in the chapter entitled xe2x80x9cthe xcex2-adrenergic receptors and G-proteinsxe2x80x9d by A. D. Strosberg in the monograph edited by the same author under the title xe2x80x9cThe Molecular Biology of Receptorsxe2x80x94Techniques and Applications of Receptor Researchxe2x80x9d and published by Ellis Horvood Ltd. Chichesterxe2x80x94Great Britain (VCH)
or also the receptors described in the publications of Brian K. Kobilka et al., Nature, Vol. 329, 75-79 of Sep. 3, 1987 or of L. J. Emorine et al., Proc. Natl. Acad. Sci. USA, vol. 84, p. 6995-6999 of, October 1987.
The invention makes possible not only the study of the mammalian membrane receptors which have been specified but also those of the receptors or polypeptides having similar structural elements which can be prepared or purified starting from standard techniques, for example
either by means of cDNAs obtained by reverse transcription of mRNA and sequenced for example according to the techniques described by Dixon, R. A. F. et al. (1986) Nature, 321, 75, Dixon, R. A. F. et al. (1987) Nature, 326, 73, Yarden, Y. et al. (1987) Proc. Natl. Acad. Sci. USA 83, 6795-6799,
or by having recourse to oligonucleotides used as hybridization probes which make it possible to isolate polynucleotides coding for receptors of the gene in question according to known techniques, including selective hybridization reactions.
These probes consist, for example, of oligonucleotide probes derived from fragments of DNA from regions showing strong homologies between the different receptors.
Probes which can be used to isolate nucleotide sequences coding for membrane receptors can also, for example, consist of conserved fragments, obtained from nucleotide sequences coding for one of the seven hydrophobic transmembrane peptide sequences shown on pages 148-149 of the monograph edited by A. D. Strosberg already cited. These different peptide sequences indicated by the roman numerals I to VII and quite similar to each other are derived from hamster xcex22-adrenergic receptors, human xcex2-adrenergic receptors, turkey xcex2-adrenergic receptors, acetyl choline muscarinic receptors from pig brain and bovine opsin.
In the case of bovine opsin, recourse can be made to the cDNA corresponding to the genomic RNA of the receptor in order to prepare one of the probes previously mentioned.
As an illustration, the following probes can be used to isolate nucleotide sequences coding for membrane receptors.
CTTGGTCAGCTTGGCCTGTGCTGATCTGGTCATGxe2x80x83xe2x80x83(transmembrane segment II)
GCTTTCATGGTCAGCTGGCTGCCCTACxe2x80x83xe2x80x83(transmembrane segment VI)
Sets of probes which can be represented by the following general formulae can also be used:
transmembrane II sets:
5xe2x80x2X1X2TX3X4TX5X6X7CX8TGGCX9X10X11X12GCTGAX13CTX14X15TX16ATG3xe2x80x2
xe2x80x83in which X1 to X16 have the following meanings, which can be varied independently in each case:
X1 is C or T
X2 is T or A
X3 is G or C
X4 is G or T
X5 is C or G
X6 is A or T
X7 is G or C
X8 is T or C
X9 is C or G
X10 is T or G
X11 is G or T
X12 is T or G
X13 is T or C
X14 is G or C
X15 is G or T
X16 is C or G
and transmembrane VI sets;
5xe2x80x2GCY1TTCATY2Y3TY4Y5Y6CTGGY7TY8CCY9TY10C3xe2x80x2
xe2x80x83in which Y1 to Y10 are independently assigned the following meanings:
Y1 is T or G
Y2 is G or C
Y3 is G or C
Y4 is C or G
Y5 is A or T
Y6 is G or C
Y7 is C or G
Y8 is G or C
Y9 is C or G
Y10 is A or T
In order to isolate nucleotide sequences presumed to code for a membrane receptor, use will preferably be made of two or more probe sequences such as those defined above chosen for their relatedness to different regions of the nucleotide sequences presenting a set of sequences in part corresponding to the seven transmembrane segments described above. In particular, use will be made of probes of the types described above with their respective complementary sequences.
Recourse may also be made to sets of these probes.
It can be seen that the procedure according to the invention makes it possible to measure for each membrane receptor expressed at the surface of a unicellular organism, in particular bacteria, the degrees of affinity of different molecules presumed to behave as ligands for these receptors. Moreover, these ligands can also be used to locate within different transformed colonies a large number of fragments of nucleic acid of various origins (the so-called xe2x80x9cshot gunxe2x80x9d approach), including fragments coding for these mammalian polypeptides. This will lead to the detection of those colonies which, on account of the expression of a sequence coding for the mammalian membrane receptor, form a bond with the corresponding ligand.
Conversely, the invention also makes possible the study of the affinity of various types of molecules for a specific membrane receptor consisting of the expression of a corresponding insert in a vector modified under the conditions defined above and incorporated in a cell host in which this vector can be replicated.
Similarly, the invention makes it possible to compare the mutual affinities of a specific receptor and different molecules possessing elements of structure in common with different mammalian membrane receptors, hence the possibility of classifying the receptors in terms of order of affinity. Any suitable ligand may be used in these comparative assays. As examples of ligands mention should be made of pindolols, iodocyano-pindolols, propanolols, alprenolols, etc . . . Reference may also be made to the monograph edited by A. D. Strosberg, in which many other agonists and antagonists of membrane receptors corresponding to the definitions of the invention are identified.
In one of these preferred applications, the invention thus relates to cell cultures composed of unicellular cells especially bacteria, containing vectors which can replicate within these organisms, these vectors meeting the conditions indicated above and containing an insert coding for a mammalian membrane receptor in compliance with the definitions indicated above, the expression of these inserts being inducible or, having been induced, leading to the transport of the corresponding membrane receptors to the membrane, depending on the nature of the promoter used.
In a preferred embodiment of the invention, the modified vector is characterized in that the insert codes for a sequence of amino acids contained in a mammalian receptor of the kind indicated above containing in its structure hydrophobic transmembrane segments inserted between extra- and intra-cellular loops, a carboxyl-terminal intra-cellular segment and an amino-terminal extracellular segment and binding sites constituted by hydrophilic regions formed in the membrane.
For the construction of the vector according to the invention, an inducible promoter is advantageously used. Such a promoter can be regulated and is only functional after induction by a biochemical molecule or by a physical treatment (treatment by temperature, ultraviolet rays, etc.). The use of such an inducible promoter enables the nucleotide sequence contained in the vector to be expressed only under conditions specified beforehand by the investigator. In this manner, damage to the cells of the transformed host culture by the modified vector is avoided, each time that the foreign receptor is to be expressed in the cell and transported into the membrane the host cell is exposed to a certain degree of toxicity.
In accordance with another embodiment of the vector according to the invention, the insert coding for the receptor can be placed downstream from a xe2x80x9cleader sequencexe2x80x9d or xe2x80x9csignal sequencexe2x80x9d and in phase with the latter, in order to form a transcription unit, this xe2x80x9cleaderxe2x80x9d sequence thus being able to facilitate the transport of the product of expression of the insert to the membrane of the unicellular host. This xe2x80x9cleaderxe2x80x9d sequence usually codes for a N-terminal sequence of a precursor of a protein of the unicellular host normally transported to the membrane of the cell, this N-terminal peptide normally being cleaved when the corresponding natural protein has been transported to the membrane of the unicellular host. Similarly, the fusion gene between this xe2x80x9cleaderxe2x80x9d or signal sequence and the insert should normally be expressed in the form of a hybrid protein cleaved downstream from the signal sequence when the receptor has been transported to the membrane of the unicellular host.
As an alternative, the insert is fused to a sequence coding for all or a part of a hydrophilic protein (for example xcex2-galactosidase), this fusion insuring that the hybrid protein, the product of the expression of the insert and the sequence coding for the hydrophilic protein, remains soluble during the transport of the receptor resulting from the expression of the insert in the unicellular host into the membrane of this latter.
The invention also relates to unicellular organisms themselves transformed by a modified vector such as that described above, and more particularly to cultures of unicellular organisms carrying on their surface sites or determinants recognized by specific ligands for the corresponding mammalian membrane receptors.
In order to implement the invention and obtain the expression of the nucleotide sequence corresponding to the selected protein, use should preferably be made of a unicellular organism selected from among bacteria, yeasts or any other unicellular organism which can accept the modified vector according to the invention and allow both its replication as well as the expression of the selected protein and its transport to the surface of the cell.
The invention also relates to a procedure for the production of unicellular organisms containing, exposed at their surface, sites characteristic of a mammalian membrane receptor, this procedure being characterized in that it comprises:
a/ the transformation of a unicellular organism by a vector which can replicate in this organism, modified beforehand so that it contains a promoter recognized by the polymerases of the unicellular host and functional in the latter and an insert placed under the control of this promoter and coding for a sequence of amino acids usually contained in a mammalian membrane receptor such as that described above.
b/ the selection of transformed unicellular organisms in which the above-mentioned insert is expressed or can be expressed in a form such that its structure comprises one or more sites exposed at the exterior of the cell membranes and is capable of being recognized by a corresponding ligand. As an alternative, the detection of expression can be performed by having recourse to specific antibodies, in particular monoclonal antibodies, which specifically recognize these same sites.
When the unicellular organism is transformed by a modified vector containing an inducible promoter upstream from the nucleotide sequence to be expressed, a step intermediate between the steps a/ and b/ cited earlier is necessary, this step consisting of the induction of the said promoter, in particular as a result of the action of a suitable substance (for example IPTG (isopropyl-D-thiogalactopyranoside) in the case of the promoter of the lactose operon and tryptophan and in the case of the promoter of the tryptophan operon) added to the culture medium when induction is desired.
The present invention also relates to a procedure for the detection of the capacity of a molecule to behave as a ligand for a membrane receptor, this procedure comprising:
a/ the placing in contact of the molecule with a unicellular organism previously transformed by a vector, itself modified by an insert coding for this membrane receptor, this organism carrying on its surface one or more specific sites of this receptor, where appropriate after induction of the expression of this insert, the placing in contact being performed under conditions leading to the formation of a bond between at least one of these specific sites and the molecule provided that it has been shown actually to possess an affinity for this receptor.
b/ the detection of the possible formation of a complex of the ligand-receptor type.
The conditions making possible the formation of the above-mentioned bond usually correspond to those conditions under which such bonds are formed between natural receptors and the ligands concerned.
The invention also relates to a procedure for studying the affinity of a mammalian receptor belonging to the class of those which are coupled to a G protein for one or more defined ligands, characterized by:
the transformation of a unicellular culture with a vector, in particular a plasmid or a phage into which has previously been incorporated a sequence of nucleotides coding for the polypeptide contained in this mammalian receptor under the control of regulatory elements, in particular a promoter, making possible the expression of the said nucleotide sequence in the unicellular culture used:
the culture of the transformed unicellular organisms under conditions which permit the expression of the insert,
the placing in contact of these unicellular organisms with specific ligands,
the detection of a specific affinity reaction between the transformed unicellular organism and the specific ligands.
The invention also relates to a kit for the detection of the possible affinity of a ligand for a specific mammalian membrane receptor, comprising;
a culture of unicellular organisms transformed by modified vector such as that described in the preceding pages in which the insert codes for an above-mentioned membrane receptor,
physical or chemical agents to induce the expression of the nucleotide sequence contained in the modified vector when the promoter placed upstream from this sequence is a promoter inducible by these physical or chemical agents,
one or more control ligands having defined affinities for the membrane receptor,
chemical or physical agents for the characterization of the biological activity of the protein.
The invention thus furnishes agents which also make possible the study of the behavior of functional proteins as receptors as a consequence of the exposure of their specific sites at the surface of unicellular organisms when the latter have been subjected to prior transformation by vectors containing an insert coding for the said functional protein. The invention also makes possible the detection or verification of the affinity of a protein as a receptor for a specific ligand. It also makes possible the study of the specificity of bonding of these receptors to different ligands selected for their importance, for example as regards future types of therapy.
A particular advantage of the invention lies in the substitution of the receptors, the purification of which is extremely difficult and the time of storage minimal, by cells expressing, or capable of expressing at their surfaces the sites characteristic of receptors which can be recognized by the ligands under study. These cells can be stored easily, particularly when the modified vector comprises a promoter which can be induced when it is required. Owing to their capacity to be cultivated, the cells enable the specific sites of these receptors to be regenerated in almost unlimited quantities on demand.
The cultures of unicellular organisms transformed by a modified vector previously described can be stored until they are required for use by placing them in particular in a 20% final glycerol solution at xe2x88x9270xc2x0 C. They may also be stored in the form of spores.
The invention lends itself to many applications. Some of them will be mentioned below, as examples.
Particularly interesting applications in the areas of therapy consist of the study of the capacity of novel xcex2-blocking substances capable of acting at the level of the xcex2-adrenergic receptors, in particular xcex21-adrenergic (in the heart) or xcex22-adrenergic (in the lung) receptors.
It has been observed that many so-called xcex2-blocking substances bind to both xcex21 and xcex22-adrenergic receptors. Certain types of therapy require, however, the use of medicines specific for one or other of these receptors and thus implies the determination of subtypes of receptors capable of binding to specific ligands. The invention makes it possible to prepare unicellular cultures capable of facilitating the screening of ligands according to the types of activities shown for different types of xcex2-adrenergic receptors: it thus makes it easy to determine the pharmacological and therapeutic specificities of medicines under study.
The invention also provides simple agents for the study of the effects of modifications introduced into the receptors with respect to their interactions with chemical substances, in particular with ligands known for their ability to bind to these receptors in their unmodified forms.
Other properties and advantages of the invention will become apparent in the description which follows and in the examples, and in particular in relation to the drawings and the table in which: